PHYTIC ACID REDUCTION IN WHCLE WHEAT FLOUR DOUGES BY pH ADJUSTMENT OR WITH SPROUTED WHEAT ADDITION A Thesis Presented to the Department of Food Science and Nutrition Brigham Young University In Partial Fullfiliment of the Requirements for the Decree Master of Science by Richard Alan Mayfield December 1982This Thesis, by Richard Alan Mayfield, is accepted in its Present form by the Department of Food Science and Nutrition of Brigham Young University as satisfying the thesis requirement for the degree of Master of Science. {Aobn Bal, domneed/< Committee Chainnan bog 2 burret Albert E. Purcell, Committee Menber f sma LB Clayton S. Huber, Departwent Chairman iiList of Tables eee eee eee List of Illustrations»... 6. + Acknowledgements © 6 eee ee ee eee ee eee Literature Reviews... eee ee Phytic Acid. ee ee ee eee Bioavailability of Divalent Minerals Phytic Acid Reduction . . . Phytic Acid Determination Experimental Procedure... 1. Experimental Design... « WheatS see eee eee Whole Wheat Flour... Wheat Sprouting... 6. Whole Wheat Flours. - eee ee eee Sprouted Wheat ee eee Whole Wheat Dough... 6. Statistics -.. 1... iii vi vii viii wee 10 10 lo u B B 18 18 20 20 20Conclusions... ee ee ee eee beeen ee OM Procedure for Flour pl Determination ....... 36 E Procedure for Phytic Acid Determination. ....+ 37 Procedure for Sprouting Wheat... eee eee ee 40 Wheat Cultivars, Their Sources and Seed Information 41 Linear Statistical Model Used for Analysis of Variance of Reduction of Phytic Acid Data... -..+.. 42 Literature Cited «eee eee ee ee eee ee AB ivLIST OF TABLES DOUGH PREPARATION PREPARATION SCHEDULE DOUGH INGREDIENTS FOR EXCH CULITIVAR GRADING FACTORS MOISTURE, PHYTIC ACID, AND pH CF WHOLE WHEAT FLOURS WHEAT SPROUTING RESULTS AND CALCULATIONS PHYTIC ACID LEVELS OF WHOLE WHEAT DOUGHS ANALYSIS OF VARIANCE TABLE FOR REDUCTION OF PHYTIC ACID PAIRWISE COMPARISONS OF THE MEANS FOR TIMES AND METHODSA 5. Myo-inositol 1,2,3,4/5/6 Hexakis (Dihydrogen phosphate) Phytic Acid Method 1: Method 2: Method 3: Method 4: Decrease of Phytic Acid Following Adjustment to pit 5.2 with 1.0 M Acetic Acid Decrease of Phytic Acid Following adjustment to pil 5.2 with 0.3 M Citric Acid Decrease of Phytic Acid Following Addition of 1% 24-hour Sprouts Decrease of Phytic Acid Follwoing Addition of 18 48-hour Sprouts viACKNCWLEDGEMENTS, The use of the laboratory facilities of Deseret Miils and Elevators, Kaysville, Utah was very helpful to the completion of this work. Special thanks is given to Dr. John Hal Johnson and Dr. Albert E. Purcell for their guidance and assistance. Gratitude is expressed to Welles Cannon for his continual encouragement and support. Miss Patty Calder has helped greatly in preparing the final copy of this manuscript. Finally, without the sustaining support and help of my wife, Mava, my daughters Sonya, Kristi, Alicia and ay parents this effort would not have reached its completion. viiINTRODUCTION The growing trend in this country of increasing use of whole wheat flour, along with the continued use of whole wheat flour by major segnents of the world population, has focused attention on the Problem of lowered bioavailability of divalent minerals due to phytic acid. This nutritonal problem has suggested the need for ways to reduce or eliminate phytic acid from whole wheat flour doughs. ‘The purpose of this study was to evaluate proposed methods of phytic acid reduction in whole wheat flour doughs. The first method was based on the addition of acetic or citric acid to obtain the optimm PA of phytase in the doughs. The second method involved the addition of sprouted wheat to increase phytase activity in the doughs. viiiEHYTIC ACID Phytic acid has been the subject of study since it was first detected in 1872 by Pfeffer. Bven though early chemical studies were done it was not until 1969 that an exact structure (Figure 1) was confirmed (Johnson and Tate, 1969). The chemical designation of phytic acid is myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate). The calciummegnesium salt of phytic acid is termed Phytin and phytate indicates the mono to dodeca anion of phytic acid. Phytic acid is found in a variety of foods. Averill and King (1926) list 57 foodstuffs (grains, flours, soybeans and nuts) with phytin contents renging from 0.66% to 3.338. Maga (1982) in his review of phytate lists cereal grains and oilseeds which contain phytate. Wheat is one of these foods containing phytic acid. Averill and King (1926) list three cultivars of wheat ranging in phytic acid level from 1.16% to 1.36%. Lolas et al. (1976) reported 38 cultivars of wheat ranging from 0,628 to 1.32% phytic acid content. A more complete study by wheat cultivar and area grown shows cultivars grown in midwestern states to be higher in phytic acid content than western wheats (Witmer, 1981). This study reported a range of phytic acid values from 570 mg (0.578) to 1346 mg (1-358) per 100 om ofFigure 1. Myo-Inositol 1, 2, 3, 4, 5, 6 Hexakis (Dihydrogen Phosphate) Phytic Acid (Oberleas, 1971).wheat. The same cultivar (Centurk) grown in three different states also had a wide range of values from 570 mg (0.578) to 963 xq (0.968) per 100 gm of wheat. In the development of the wheat kemel, phytic acid is fotmed by inositol phosphorylation. Asada et ai. (1969) proposed the hypothesis that phytic acid in cereal geains does not occur in a sequential fashion through phosphorylated inositol, but with a hypothetical phosphorylated incsitol derivative. This hypothesis has inositol phosphorylated by @ phosphate donor to inositol monophosphate which is then complexed by an unknown reagent X to form a phosrhorylated inositol derivative. This derivative is then Phosphorylated until all six positions on the inositol have phosphates attached. The derivative then releases the phytic acid. The role of phytic acid in wheat suggested by Williams (1970) is the storage of phosghorus for future plant use and the slowing of metabolism prior to dormancy by the rapid formation of phytic acid neat maturity. Phytic acid is shown to contain from about 508 to 80% of the total phosphorus in wheat (Bassiri and Nahapetian, 1977; Lolas et al., 1976; Nahapetian and Bassiri, 1975). This high level of phosphorus suggests a storage role for phytic acid. ‘BIOAVAILABILITY OF DIVALENT MINERALS Phytic acid has been suggested 2s the cause of lowered bioavailability of various divalent minerals. Various investigators pave reported lowered bioavailability of calcium due to phytic acid(Hoff-Jorgensen et al., 1946; Bruce and Callow, 1934). Haghshenas et al. (1972) attributed the occurrence of iron-deficiency anemia of Iranians to high phytate levels from their main dietary staple of unleavened whole wheat bread, This effect occurred despite a hish iron content in their diet. In the 1950's zinc bioavailability for laboratory animals was being investigated due to zinc deficiency problems. O'dell and Savage (1960) reported lowered zinc bioavailability in studies with phytic acid added to various feeds. In studies with rats Oberleas et al, (1966) indicated decreased bioavailability of zinc due to phytate. A further study by O'dell et al. (1972) in evaluation of zinc availability from plant and animal sources indicated that phytic acid appeared to cause a lower availability of zinc from plant sources. More recently Franz et al. (1980) and Witmer (1981) both reported an inverse relationship between phytic acid and bioavailability of zinc. Finally, Oberleas et al. (1966) also indicated an increased lowering of zinc bioavailability in the presence of excess calcium, In one study Reinhold (1975) suggested that phytic acid my be a possible cause of human zine deficiency in Iran. However, Reinhold et al. (1975) reported that fiber and not phytic acid was the primary cause of lowered divalent mineral bioavailability. In contrast to those results, Davis et al. (1977) concluded that phytate and net fiber was the major determinant of zinc bioavailability. Although Reinhold et al. (1976) concluded that dietary fiber was a major factor affecting zinc availability, Daviset al. (1977) found the correlations between zine and phosphorus were closer than between zinc and fiber. Since most of the phosphorus was present as phytate it would sucgest rhytate was rore important than fiber. PHYTASE Phytase is the enzyme which catalyses the hydrolysis of phytic acid to inositol and phosphate. Peers (1953) reported that phytase purified from whole wheat flours had a pH optimum of 5.15 and an optimum temperature of 55°C. The Michaelis constant for phytase was 0,3 X 103M. The usual heavy metal salts completely inhibit the enzyme, And hard wheats have a higher acitivty than soft wheats. Nagai and Funahasi (1962) indicated that phytase of wheat bran was not a phytin-specific phosphatase but the same as an enzyme known as plant nonspecific acid phosphomoncesterase. Later studies indicate that phytase from wheat bran can be separated into two fractions F, and P, by DEAE-cellulose (Lim and Tate, 1971). these fractions yield different substrate degradation patterns and F) is activated by lysolecithin, Finally the results of Lim and Tate (1973) suggest that a variety of phytases are present in biological systems. BEXTIC ACID REDUCTION During World War IT the lowered absorption of calcium due to Phytic acid was of great concern because of the higher levels of phytic acid in flour due to inigher extraction rates with greater ‘ran content. Pringle and Moran (1942) studied the effects of bakingon phytic acid and concluded that phytase in flour caused the breakdown of some phytic acid during breadmaking. The longer the fermentation time the greater the destruction of phytic acid. DeLange et al. (1961) studied factors influencing phytic acid hydrolysis in bread baking and reported that longer fermentation tines of three, four or five hours increased phytic acid reduction, though their results indicated slight effects after three hours. The addition of acid, acetic and citric acid, increased phytic acid reduction. The addition of calcium acetate, however, inhibited greatly the reduction of phytic acid. Ter-Sarkissian et al. (1974) in their study of Iranian breads show that modifying baking methods can reduce phytic acid content. ‘hey point out that longer fermentation tines are successful in reducing phytic acid level but produce an undesirable result in some of the sour dough breads and, therefore, recommend a lover extraction flour in order to reduce phytic acid content. Reinhold (2975) proposed that the nutritional value of Iranian bread could be improved if the traditional unleavened bread were leavened and made from a lower extraction flour. The result of these changes would be lower phytic acid content. The commercial removal of phytic acid from soy protein uses a different aspect of phytic acid chemistry. Hartman (1979) reported a Process of solubilization, pH adjustment to 11.6 to precipitate the phytate and centrifugation to remove it. Finally the high pil of the soy protein dispersion is neutralized. This procedure lowered the phytate content from 2.68 to 0.18 for a typical comercial soyProtein isolate. Farland and Harland (1980) reduced phytate content in three breads (rye, white and whole wheat) by doubling the yeast in each recipe and extending the fermentation time. Knorr et al. (1981) used commercial phytase or phosphatase in order to reduce phytate levels to 1/8 and 1/12 of initial values. They reported the continued decrease in phytate to a level of only 0.6 m¢/100 om of bread after 96 hours of storage following baking. Finally, Tangkongchitr et al. (1981) followed the loss of phytate through bread-making and concluded that the principal barrier to reduction of phytate in bread-neking is the insolubility of magnesium phytate in dough. A later report by Tangkongchitr et al. (1982) indicated that the ingolubility o£ magnesium phytate could be decreased at pa 5 where the limiting factor appeared to be the level of phytase. PHYTIC ACID DETERMINATION Beuber and Stadler (1914) developed the method upon which most procedures for phytic acid determination are based, This method uses the fact that phytate in dilute acid forms an insoluble complex with ferric ions. The phytate extract was titrated with standardized ferric chloride solution using ammonium thiocyanate as an indicator. The end point was reached when a flesh=pink color persisted fer five minutes. This color was produced by an excess of ferric ion which formed ferric thiocyanate. The major disadvantage with this method is the lack of a sharp end-point due to the ferric phytate percipitate. Young (1936) modified the method by adding a fixedamount of standard ferric chloride and measured the excess unprecipitated ferric ion colorimetrically. Others have modified this basic method (Earley, 19447 Debange, 1961). These methods based on Heuber and Stadler (1914) have been @ivided into "direct" and “indirect" analysis of phytic acid (Senotus and Schwinmer, 1962). The "direct" methods were based on the determination cf phosphorus content of carefully precipitated ferric phytate after hydrolysis. The "indirect" methods measure the unprecipitated iron or the portion of a standardized iron solution precipitated by phytic acid in dilute acid. Generally, the "indirect" methods are more convenient (Oberleas, 1971). Comparison of these methods have been made by other authors who report. wide variation in results. Marrese, et al. (1961) conpared three methods of phytin determination and showed wide differences in results. They concluded that investigations about phytin are dependent upon an understanding of what phytin is and with what confidence it can be measured. A comparison of four procedures for phytic acid determination by Makower (1970) also denonstrated a wide variation in results. She concluded that determination of iron in ferric phytate via ferric hydroxide gives results comparable to those obtained in determinations from wet-ashed ferric phytate for phosphorus or iron. The residual iron method is more variable particularly with small amounts of piytic acid and is subject to greater error. Wheeler and Ferrel (1971) in their study of phytic acid determinaticn in wheat and wheat fractions also encountered this variation in results.A medification of the wet-ashed ferric phytate method was eveloped by Harland an@ Cberleas (1977) in which the phytate is concentrated on an anion-exchange resin. The phytate concentrated on the anion-exhange resin is stripped of contaminating inorganic phosphate with a weak sodium chloride solution. The phytate is then eluted with a stronger sodium chloride solution. The final eluant fraction is then digested and the phosphate is measured. A modification of this method proposed by Latta and Eskin (1980) uses the anion-exchange resin to concentrate the phytate and remove inorganic phosphates however, instead of digesting the final eluant fraction it is mixed with Wade reagent and the amount of phytate is colorimetrically determined. Wade reagent (0.038 ferric chloride - hydrate and 0.3% sulfosalicylic acid in distilled water) has a pink color due to the reaction between ferric ion and sulfosalicylic acid. When Wade reagent is mixed with phytate the ferric ion ts bound to the phytate and unable to react with the sulfosalicylic acid, which results in a decrease in the intensity of the pink color. This intensity decrease is measurable and proportional to the amount of phytate present. This nethod, even though it is simple and rapid, has resulted in low phytate values in comparison to earlier nethods. Ellis and Morris (1982) in their comparison of this method and the ircn precipitation method concluded that interfering substances in the acid extract resulted in lover phytate levels. They found that this effect is minimized or eliminated by Precipitating the phytate and regenereting the purified phytate before adding the sample to the anion-exchange resin.EXPERIMENTAL DESIGN ‘The purpose of this study was to evaluate proposed methods of phytic acid reduction in whole wheat flour doughs from two cultivars of Utah wheat. Acetic and citric acids were added to doughs to obtain a flour pH of 5.2. This was done to reach the approximate optimum p& of phytase. Doughs made with 18 sprouted wheat added were also prepared. The sprouted wheat was added to determine if 24-hour or 48-hour sprouting would increase phytase activity. The doughs prepared by these methods were allowed to set for zero, two and four hours. the phytic acié levels of the flours and the doughs were determined and the relative effectiveness of the methods were compared vsing these resytts, The experimental design was reviewed by the Center for Statistical Research at Brigham Young University prior to starting to insure statistical balance. Three replications of each cultivar, method and time were prepared and each replication was analyzed for phytic acid in duplicate. MERATS ‘Iwo cultivars of wheat from Utah were obtained for this study (dansel and Nanning). Appendix D contains the sources of these wheats and the seed information supplied. The wheats were graded in accordance with the standards established by the United States Department of Agriculture (1978).wd The wheats were also analyzed for protein, using the Kjeldahl method, and moisture using the dielectric method for whole kernel moisture determination. ‘WEOLE WHEAT FLOOR The two cultivars of wheat were divided into two 25-pound samples by using a Boerner sample divider. ‘These two samples were then ground into flour using a home flour mill.* The milled flour was then placed in plastic one-gallon jars and frozen at 0°F until the doughs were prepared. Four 3.000 gm samples of flour from each cultivar were weighed into aluminum cups and dried at 95°C at a vacuum of 24 inches of mercury in an oven for three hours. Samples were allowed to cool in a desiccator, weighed and the moisture calculated from the weight joss. The pH's of the whole wheat flours were determined using a modification of the AACC method 02-52 (American Association of Cereal Chenists, 1976). Four samples of each flour were placed in beakers and recently boiled distilled water, cooled to 25°C, was added. The water and flour mixture was agitated at 25°C for a period of time and then allowed to settle. The pil of the supematent liquid above the flour was read using a pi meter with a combination glass electrode (Appendix A). ‘Magic Mill II, Magic Mil] Co., Salt Lake City, Utah. A micronizing mill.12 Flour samples were analyzed in quadruplicate for phytic acid by the method of Latta and Eskin (1980) as modified by Ellis and Morris (1982), ‘This procedure measures phytic acid colormetrically using the Wade Reagent (0.038 FeCl,~6H,0 in 0.38 sulfosalicylic acid). The phytic acid wes extracted from the flour with 2.48 HCl solution. Excess ferric chloride solution was added to the extract and ferric phytate was precipitated. The precipitate was washed and sodium hyéroxide was added to form ferric hydroxide and sodium phytate. The sodium phytate solution was then passed through an anion exchange column. the column was made with 0.5 gm of 200-400 mesh Dowex 1 X 8 resin, The interfering phosphates were removed from the column using 0.1 M sodium chloride. Finally, the phytate was eluted from the colum using 0.7 sodium chloride. This eluant fraction was mixed with Wade Reagent and the absorbance of the solution read at 480 nm. The ug/ml of phytic acid was determined by a least squares linear regression of the standard soluticn calibration data (Appendix B). Reagent grade concentrated acetic and crystalline citric acids were diluted to form 1.0 M acetic acid and 0.3 M citric acid. These solutions were used to lower the pil of the whole wheat dough to a pit of 5.2. In order to determine the amount of acid needed to lower the pA of the dough, samples of each flour in quadruplicate were used to Getermine the pi of the flour. ‘the acids were added, using a buret, to these supernatants until the pi of the liquid had been lowered to 5.2,13 ‘The amount of acid added was averaged for the four samples of each wheat and this value was used to determine the total amount of acid needed for each whole wheat flour. ‘MEEAT SPROUTING In order to provide the necessary sprouted wheat for this study, each cultivar of wheat was sprouted for 24 and 48 hours. The whole wheat was sprouted by soaking the wheat in two to three cups of warm water for eight to nine hours; then the wheat wag rinsed and allowed to sit in @ dark location for the retaining time. ‘the sprouts were rinsed every four to six hours during the last 16 to 40 hours. The sprouts were then frozen until the doughs were prepared. Using the beginning and final weight of the sprouts, the amount necessary for 18 sprouted wheat (Gry weight) in the douchs was calculated (Appendix C). WHOLE WHEAT DOUGH ‘The doughs were prepared by cultivar, method and time as noted in ‘Table 1. Each dough sample was prepared in triplicate in accordance with the schedule in Table 2. This schedule was a random generation by computer in order to provide a statistically balanced experinent. In Method 1, flour, water and 1.0 M acetic acid were mixed to form a dough consisting of 100 gms of flour (dry matter basis) and 100 ml of Liquid. In Method 2, 0,3 M citric acid was used instead cf acetic acid. Method 3 was prepared by taking the 1% by weight of 24-hour sprouts and blending then with the water in a householdTABLE 1 DOUGH PREPARATION Dough Sample cultivar Method* Time ‘Nunber (hours) 1 Hansel 1 0 2 . 1 2 3 ® 1 4 4 . 2 0 5 . 2 2 6 * 2 4 7 . 3 0 8 . 3 2 9 " 3 4 10 " 4 0 aL * 4 2 32 * 4 4 13 Manning 1 ° 14 " 1 2 15 " 1 4 16 " 2 9 uv : 2 2 18 . 2 4 19 . 3 0 20 . 3 2 21 . 3 4 22 * 4 a 2B * 4 2 24 . 4 4 * Method 1 - 1.0 M acetic acid added to lower pH to 5.2 of * Method 2 - 0.3 M citric acid added to lower pi to 5.2 of * Method 3 - 18 24-hour sprouted wheat added to dough. * Method 4 - 18 48-hour sprouted wheat added to dough. dot co g& 14sample Preparation Sample Order Bough Order Rough Number Number L 2 37 6 2 12 38 15 3 3 39 15, 4 1 40 22 5 4 41 5 6 3 42 4 7 2B a 18 8 3 44 3 9 4 20 wv 1 i 916 blender and then mixing with the flour. Method 4 was prepared with 48-hour sprouts in place of the 24-hour sprouts. All four methods were mixed in a household mixer* until a uniform dough was formed (approximately three minutes). the doughs were then placed in plastic freeger bags and either frozen with dry ice for 1 hour time or allowed to react for either two or four hours at roan temperature (25° to 27°C). After two or four hours these doughs were also frozen with dry ice. The actual amounts of flour, water, and acid or sprouts used in each method and cultivar are shown in Table 3. ‘The determination of phytic acid in the dough was done in the same method as the flour except the weighing of the dough was done while it was still frozen for ease of handling (Appendix B). *RitchenAid Mixer Model KSA, Hobert MEg. Co., Troy Ohio.‘TABLE 3 ‘DOUGH INGREDIENTS FOR EACH Method #1 (Acetic Acid) Flour water Acid (1.0 M Acetic Acid) Method #2 (Citric Acid) Flour 109.0 gm 109.2 om Water 77.0 mk 77.3 mb Acid (0.3 M Citric Acid) M0 sl 13.5 mL Method #3 (24-Hour Sprouts) Flour 107.9 gm 108.1 gm Water 91.3 mL 91.0 mil Sprouts 1.80 gn 1.88 gm Method #4* (48-Hour Sprouts) Flour Water Sprouts + Additional weight of 48-hour sprouts was added to compensate for increased water absorbance by 48-hour sprouts.RESULTS ‘WHOLE WHEAT PLOUR The two cultivars of wheat both graded U.S. No. 1 wheat (Table 4). The two ailtivars varied by 1.28 protein and 0.4% moisture (Hansel 13.9% protein and 11-18 moisture; Manning 12.7% protein and 11.58 moisture). The whole wheat flours were very close in phytic acid level (iansel 742 mg/100 gn and Manning 745 mg/100 gm) for the cultivars (Table 5). In comparing the standard deviations of the phytic acid determination with previcusly reported values (Witmer, 1981; Latta and Bskin, 1980; Makower, 1970) these values are within the ranges reported. ‘The pH of both cultivars are a little higher than 6. ‘the moisture content of the flour was approximately 38 lower than the intact wheat kernel. This lowered moisture content is probably due to moisture Joss during flour milling. ‘The determination of the required amount of acid is based oa the amount of 1.0 M acetic acid or 0.3 M citric acid added to the flour supernatant to lover the pH to 5.2 for 10 grams of flour. These values were then multiplied by 10 to determine the amount of acid to add to the 100 grams of flour in the dough. the results were 4.4 ml of acetic acid for both cultivars and 14,0 ml of citric acia for Hansel and 13.5 mi of citric acid for Nanning.Hansel Manning Class Bard Red Winter Hard Red Winter Dockage 0.08 0.08 Protein 13.938 + 0.66 12.73% + 0.05 Moisture 1s 11.58 Test Weight 60.8 62.7 (pounds per bushel) Grade + Not 0.8. No. 1 ‘TABLE 5 Cultivar MOISTURE, PHYTIC ACID, AND pH OF WHOLE WHEAT FLOURS cultivar Moisture Phytic Acid PH 3 mg/100 gm Hansel 8.228 4 0.24 742 + 24 6.3 40.1 8.42% + 0.13 745 + 62 6.44001SERQUIED WHEAT The 24-hour sprouts had one shoot of approximately 1/8 inch long, while the 48-hour sprouts had three shoots from approximately 1/4 to 5/8 inch long (Table 6). ‘The calculations used to determine the weight of sproute added to the doughs are shown in Table 6. The end result is the addition of from 1.80 to 2.24 gus of sprouts to the dough to add 18 dry weight of sprouts. ‘WEQLE WHEAT Dony The phytic acid levels of the whole wheat doughs (method 1 (@eetic Acid) shows a rapid decrease in phytic acid levels from over 700 mg/100 gm to about 200 mj/100 om after two hours in both cultivars (Table 8, Figure 2}. The reduction rate levels out after two hours and after four hours very little additional reduction in Bhytic acid level had occurred, Method 2 (Citric Acié) has a similar pattern except the reduction at two hours is slightly greater (Figure 3). Method 3 (24-hour sprouts) had a pattem of very gradual reduction until two hours and it also ieveled aut to very little additional reduction (Figure 4). Method 4 (48-hour sprouts) for the cultivar Manning showed a similar pattem as Method 3. However, for the cultivar Hansel the pattern appears to rise up to a high of about 630 mg/100 gm at two hours and decrease to a low of about 440 mg/100 ga at four hours. statistics The data for the phytic acid levels in the flours and doughs were analyzed for statistical significance using the computerStart Weight 24-hour 75) gm 750 am 48-hour 73 ga 3 on Finish Weight 24-hour = 120 gm 124.6 gm 48-hour 145.5 gm 149° gm Initial Moisture Content 11.18 11.58 Actual Grams of Wheat 66.68 gn 66.38 gm (dry matter) Based on 75 gm Grams of Sprouts 24-hour 1.80 gn 1.88 gm 48-hour 2.18 aw 2.24 gm ‘The number of grams of sprouts to equal 2 gram dry matter is the total finish weight divided by actual grams of weight (dry matter).‘TABLE 7 PHYPIC ACID LEVELS CF WHOLE WHEAT DOUGHS Dough Sample Cultivar Method* = ‘Tine umber Bours 1 Hansel 1 0 2 . 1 2 3 " i 4 4 2 o 5 2 2 é 2 4 7 3 0 8 3 2 9 3 4 20 . 4 9 i 4 2 2 . 4 4 3 Manning 1 0 aT " 1 2 13 1 4 16 " 2 0 uv 2 2 18 2 4 19 3 oO 20 3 2 21 " 3 4 22 4 0 2B 4 2 24 " 4 4 * Method 1 - 1.0 M acetic acid added to lower pH to 5.2 * Method 2 ~ 0.3 M citric acid added to lower pH to 5.2 * wethod 3 - 18 24-hour sprouted wheat added to dough. * Method 4 - 18 48-hour sprouted wheat added to dough. Phytic Acid mg/100 gm 361+ 70 216 + 115 248 + 61 382 + 107 145 + 59 13 + 30 660 + 92 500+ 93 484 + 123 545 + 118 634 + 92 446 £56 459 + 143 220+ 23 2124 73 534 + 125 161+ 49 214 52 664 + 113 S71 + 108 562 4 71 660 + 105 526 + 182 592 + 53 of Gough. of dough.700 600 MG PER 100 GRAMS 300 PHYTIC ACID 200 TIME Figure 2. Method 1: Decrease of adjustment to pi 5.2 with 1.0 M acetic acid. wom HANSEL arm MANNING HOURS Paytic acid followingMG PER 100 GRAMS PHYTIC ACID wom HANSEL mon MANNING TIME «Hours Figure 3. Method 2: Decrease of adjustment to pH 5.2 with 0.3 M citric acid. Fiytic acid followinga HANSEL won MANNING MG PER 100 GRAMS 400 PHYTIC ACID 2 TIME Hours Figure 4, Method 3: Decrease of phytic acid follwoing addition of 18 24-hour sprouts.woe HANSEL mn MANNING MG PER 100 GRAMS on PHYTIC ACID 2 TIME Hours Figure 5. Method 4: Decrease of phytic acid following addition of 18 48-hour sprouts.a program Rummage IT updated April 23, 1982 by Del T. Scott, Melvin Ww. Carter and Gale Rex Bryce of the Statistics Department, Brigham Young University. This analysis indicated no statistically significant disterence between Nethods 1 (Acetic acié) and 2 (Citric acid) or Methods 3 (24-hour sprouts) and 4 (48-hour sprouts). However, the analysis indicated statistically significant difference between Method 1 (Acetic acid) and Methods 3 (24-hour sprouts) and 4 (48-hour sprouts) and between Method 2 (Citric acid) and between Methods 3 (24-hour sprouts) and 4 (48-hour sprouts). Finally, the analysis indicated no statistically significant difference between two and four hours of reaction time. The linear statistial model for this analysis and the key to the model are shown in Appendix BE. Tables 8 and 9 are an analaysis of variance table for reduction of phytic acid and paired comparisons of the means of the time and nethods.TABLE 8 ANALYSIS OF VARIANCE TABLE FOR REDUCTION OF PHYPIC ACID Source of Error Degree Sum Mean F Variation Tem of of Square Ratio Freedom Squares V (cultivar) R 1 80586.8 80588.8 — 4.433* M (method) R 3 3133742.9 10445681.0 57.464 VEN R 3 13402.2 4467.4 0.246 T (time) R 2 876755,3 438377.6 24.116* VxT R 2 5€339.1 29169.6 1.605 MxXT R 6 262816.7 43802.8 — 2.410* VENKE R 6 124291 ..2 20715.2 1.140 R (replication) 48 672551.7 18178.2 © 5,788* Ecror 72 226121.5 3140.6 ‘TOTAL 143 5648609.3 Sinple Resquared 960 Reequared adjusted .920 *Siqnificant at 0.05‘TABLE 9 PAIRWISE COMPARISONS OF ‘THE MEANS FOR TIMES AND METHODS Pairs Difference Standard Tvalue of Means Error u-m 161.458 27,521, 5.867" n-% 169.313 27.521 6.152* m-B 7.854 27,52. 0.285 ML - M2 20.674 31.779 0.681 mM - 93 ~287.667 31.779 3.052" m+ M4 -280.938 31-779 8.840* m2 - 3 -308.340 31-779 9.703% M2 - M4 301-611. 31.779 9.491* 1 - ME 6.729 31.779 0.212 ‘*Significant at 0.05 T = 0 hours B= 2 hours hours 1] = wethod 1 - 1.0 # acetic acid added to lover wil to 5.2 ‘of dough: MW, = Method 2 - 0.3 M citric acid added to lower pH to 5.2 ‘of dough. = Method 3 - 18 24-hour sprouted wheat added to dough. iy = Method 4 - 1¢ 48-hour sprouted wheat added to dough. Refer to Appendix E page 42 for linear statistical modelDISCUSSION The addition of acid to lower the pl to 5.2 (Methods 1 (Acetic acid) and 2 (Citric acid)) resulted in a similar pattem of reduction in phytic acid. With both acids a sharp reduction in Bhytic acid occurred during two hours, followed by very little additional reduction for four hours of reaction time (Figures 2 end 3). These results are similar to those reported by Harland and Harland (1980), Delange et al- (1961) and Tangkongchitir et al. (1981, 1982). This reduction in phytic acid is probably due to erzymatic activity of the phytase present n the doughs. The apparent rate reduction of enzymatic activity after two hours may have at least two possible causes. Tangkongchitir, et al. (1981) concluded that this loss of enzymatic activity was due to the insolubility of magnesiumm phytate in the dough. Tangkongchitir et al. (1982) showed that lowering the PA to 5.0 seems to overcone this insolubility problem. However, Oberleas (1962) suggested that in his studies of precipitation of phytate with divalent minerals, most of the phytate in wheat would be soluble at the pH of 6.2 to 6.6 (the average pi of whole wheat flour). Therefore, the insolubility of magnesium phytate would not explain this rate redsction of enzymatic activity. The second possible cause is that the phytase may be inhibited. the pecbable reaction is the phytate being hydrolyzed by phytase to either inositol and inorganic phosphate, or a lesser phosphorylated inositol and inorganic phosphate. These products of enzymatic activity, inositol, inorganic phosphate or a lesser phosrhorylatedal inositol probably inhibit the phytase. This appears to be the most likely mechanism. ‘The aGdition of acid lovers the pH to 5.2, which optimizes the phytese activity within the dough. This activity continues until a build-up of these inhibiting agents occurs. This build-up is probably localized due to a loss of mbility of the Phytate within the dough as the dough structure forms. This localized inhibition and loss of mobility would explain the same pattem of Little additional reduction in phytate between two and four hours with the addition of wheat sprouted for 24 and 48 hours. The pH of the sprouted wheat doughs would be that of ordinary whole wheat flour (pH 6-2 to 6.6) wiich is higher than the optimm pil of 3.2 for the phytase and this would result in a lower level of phytase activity and explains the smaller overall reduction in phytic acid. Delange et al. (1961) used 100% extraction flour and added vinegar and citric acid to lower the crumb pH to approximately pH 5.7 to pH 6.1 with fermentation times of three, four and five hours. The results indicated the greatest reduction of phytic acid with the addition of both vinegar and citric acid. The addition of both resulted in 2 77% to 80% reduction of phytic acid and a PH of approximately 5.7. The Delange study also shows little additional reduction after three hours of fermentation. The results match closely the results in this study; Towever, due to the higher pli's, not a5 mich reduction of phytic acid occurred in the Detange study. Tt is interesting to note that both acid additions caused a significant drop in phytic acid in doughs prepared without reaction time (0 hours}. The mixing of the dough and lowering of the pH to32 5.2, without a reaction time after mixing, resulted in increased Bhytase activity which cavsed considerable reduction of phytic acid before the dough sample was cooled below the inactivation temperature of the enzyme. This same drop is noted in the sprouted wheat doughs at a smaller scale. The time required to show an apparent rate reduction in emymatic activity was measured at two hours in this study; howver, it should be noted that no data were collected between zero and two hours, which means the rate reduction could have occurred at any time between zero and two hours after mixing. Further study in this rate reduction of enzymatic activity could attempt to increase mobility within the doughs possibly by remixing the doughs after two hours of reaction time or determining the inhibiting agents and mixing them in the doughs to observe their effect on phytic acid reduction. The acid addition reduces phyti¢ acid and in turn may overcone the adverse effect on divalent minerals. This reduction, however, may not ke enough to completely overcome the adverse effect of the phytic acid in the doughs. Addition of wheats sprouted for 24 or 48 hours shows results that would te expected for enzyme action with no additional phytace activity being produced during the first 48 hours of germination. These results are in acreenent with the reported decomposition of Phytic acid in wheat during germination by Mihailovic et al. (1965). They reported that the majority of phytic acid decomposition occurs from the third to the seventh day of germination. Apparently before the third day of germination little additional phytase activity is33 produced. The addition of wheats sprouted for longer than 72 hours (three days) would possibly increase phytic acid reduction; however, this increased germination could cause undesireable results in the dough structure because of increased amylase activity. The unexpected results of an apparent rise in phytic acid level for the addition of 18 48-hour sprouts (Method 4, Figure 5) with cultivar Hansel between zero hours to two hours reaction tine is not as significant as it appears if it is noted that the standard deviations of the results place both results within a standard deviation of one another. Close examination of the standard deviations will also note that the apparent differences in the zero reaction tine phytic acid levels for the cultivar Nanning with acid addition are also within a standard deviation of each other. This results in ne significant difference between the acid addition methods (Method 1 and 2). The use of room temperature (25° to 27°C) for reaction time of the doughs instead of the optimm temperature of 55°C for phytase activity wes due to the use of room temperature or slightly higher for most breadmaking. ‘The higher temperature (55°C) would possibly begin baking the dough and this could increase even further the loss of mobility within the dough. ‘The use of both acid addition and sprouted wheat together is a possible method for phytic acid reduction. The effect would probably be reduction of phytic acid; however, the need to increase sprouting ‘ine to longer than 72 hours to increase phytase activity would also increase amylase activity and cause possible undesirable effects on the dough structure.34 CONCLUSIONS ‘the addition of acid to whole wheat doughs and allowing the doughs to set for two hours resulted in the greatest significant reduction of phytic acié in whole wheat doughs. ‘The use of 24-hour and 48-hour sprouts did not increase the phytic acid reduction rate above that expected from natural phytase present in the flour. Pinally, the greatest reduction in phytic acid appears to occur in the first two hours after the doughs are prepared.35 APPENDICES36 APPENDIX A Procedure 1. Place 10 ga of flour into a dry 150 ml beaker. 2. Add 100 ml of cool, recently boiled distilled water at a temperature of 25°C. 3, Agitate the flour until an even suspension free from lumps is obtained. Allow suspension to stand at 25°C for 30 minutes, agitating intermittently in such a manner as to keep the flour particles in suspension. Let stand for 10 additional minutes. 4. Using a calibrated pH meter, determine the pH of the supernatant Liquid.37 APPENDIX B Reagents 1. 2.4% (w/y)_EC1 Solution Dilute 63.7 ml of concentrated HCl in a 1000 ml volumetric flask with distilled water to the 1000 ml line. 2+ FeCl, solution in 0.68 ACL Disséive 4 gm of FeCl, - 68,0 (Mallinckrodt Reagent lumps) in 1 liver of 0.68 Hele 3. 1M Nao Dissolve 40.00 gm of NaCH (Baker Reagent Grade ACS) in a 1000 mt volumetric flask with distilled water to the 1000 ml line. 4, 0.7 M Nach Dissolve 40.91 gn of NaCl (Fisher Scientific ACS) in a 1000 ml volumetric flask with distilled water to the 1000 ml mark. 5. 0.2 M NaCl Dissolve 5.85 gn of NaCl (Fisher Scientific ACS) in a 1000 ml volumetric flask with distilled water to the 1000 ml mark. . Wade Reagent 0,038 FeCl, - 610 and 0.3% sulfosalicylic acid (Spectrum Grystat Reagent ACS) in distilled water. 7, Standard Solutions A series of standard solutions containing 5, 10, 15, 20, 25, 30, 35, 40 ng/ml of phytic acid (Sigma Chemical Company). 8. 58 (w/v) BCL solution Dilute 132.6 ml of concentrated HC] in a 1000 ml volumetric flask with distilled water to the 1000 ml mark. Procedure i. Detemmine a calibration curve for phytic acid concentration vs. absorbance. ‘Take § ml of 2 standard solution and add 2 ml of Wade Reagent solution in a 15 ml conical centrifuge tube. Mix for 5 seconds on a vortex mixer. The mixture is centrifuged for 1G minutes. Read the absorbance of the solution at 480 nm with a spectrophotneter using vater 2s a zero. Using the absorbance values and concentration levels determine the linear Beer's Law plot for the standard scluticns.4. 5. 6. 38 Weigh out $.000 gm of flour ot 10.000 gn of dough and place into a 150 ml or larger beaker. Add 100 mi of 2.4% HCl, extract phytic acid by thoroughly agitating mixture and intermittently agitating selution for at least 1 hour. ake 5 mi of extract and mix with 5 ml of Fecl, solution. Beat mixture in boiling water for 15 minutes. “Cool in ice water. Centrifuge mixture and wash precipitate with distilled water two times. Decant excess liquid from precipitate. Add 5 ml of 1.0 M NaOH solution, agitate mixture to redisolve phytic acid. Decant liquid into 25 mi volumetric flask. Wash ferric hydroxide precipitate two times with distilled water and add vashings to volumetric flask. Add distilled water to the 25 mi mark, Take 10 ml of the redisclved phytic acid solution and add to column. Colum is made with 0.5 gm of 200-400 mesh Dowex 1X 8 resin. ‘he Dowex resin was prepared for use by adding distilled water, agitating the mixture, allowing the resin to settle and pouring off the supematant to renove the finer particles. ‘This process was done three tines. Remove inorganic phosghates or other interfering agents by adding 15 ml of 0.1 N NaCl to the colum. Remove the phytic acid by adding 15 ml of 0.7 H NaCl. Catching the elutant as soot as the 15 ml of 0.7 NaCl is added to the colum. ‘The elutant is removed from under the column as soon as the level of liquid reaches the top of the resin. ‘The elutant is diluted to a volume of 25 ml with distilled water, and mixed thoroughly. Take 6 ml of mixture and add 2 mi. of Wade Reagent in a 15 mi conical centrifuge tube. Mix for 5 seconds on a vortex mixer. The mixture is centrifuged for 10 minutes. Read the absorbance of the solution at 480 rm. Regenerate the columns by adding 15 ml of 5¢ HCl solution and then add 20 ml of distilled water.39 CALCULATION Ua/nl of phytic acid determined ty least-squares linear regression of standard solution calibration data. ugém_X 25 ml X 25 mlx 100 mix 100m X md = mgof phytic acid 10 mi ¥ S ml X dry we of sample X 1000 ug 100 gm of dry sample uodml 4125 = me of phytic_acid Gry wt of sample 100 om of dry sample40 APPENDIX C Procedure 1. Weigh out 75 gm of wheat and place it into a mason jar. Attach plastic screening over end of jar with jar ring. 2. Add two to three cups of warm water to the jar at 25°C. Let wheat soak for eight to nine hours in a dark location. 3. Drain wheat and rinse in cool water once and rinse in 25°C water two or three times. Drain and place jar in a tray so that water drains from wheat and air gets to wheat. Store between rinses in dark location. 4. Rinse sprouts every four to six hours with 25°C temperature water two or three times for rest of 24 or 48 hours. Return jar to drain tray and store in dark location.APPENDIX D Wheat Cultivars, Their Sources, and Seed Information Source Quitivars Utah Crop Improvement Association Utah Agricultural Experiment station Hansel Logan, Utah 84321 Wheatland Seed, Inc. Mennning Brigham City, Utah 84302 Seed Information cultivars Hansel Manning Origin Utah Utah Lot Munber/Cert. No. UAES-2/H-10 MW 3010 Pure Seed 99.718 99,008 Inert Matter 0.298 1.008 Other Crop 0.008 0.008 Weed Seed 0.00% 0.008 Germination 94,258 Date Tested 9-32-8042 i APPENDIX E \ LINEAR STATISTICAL MODEL USED FOR ANALYSIS OF VAREANCE OF REDUCTION OF PEYTIC ACID DATA Yoga 2 OS + Vy + May + Tie + ME +E, + Woe + Reis * Beajom REY TO MODEL: i=1,2 V(cultivar) 3=1,2,3,4 M(method) = 18 24-hour sprouts (= 18 48-hour sprouts hours hours = 4 hours k=1,2,3 TCtime) sue 1,23. (replication) Ast replication 2nd replication 3rd replication epee BR RRR as Bee m=1,2 terror) st observation 2nd observation Re une Ysgjan ~ Piytic acid reduction of, the fh dbeervation of the x replication of the it cuitivar, j*Y method and k* tine ug = General mean of reduction ve = An effect due to cultivar MS = An effect due to method vais = Interaction effect between cultivar and method yy = An effect due to time VES, = Interaction effect between cultivar and time Ts, = Interaction effect between method and time Wer, sk 7 interaction effect between cultivar, method and time R= An effect due to replication, also error term for preceeding effects B= Error term.43 LITERATURE CITED44 LITERATURE CLTED American Association of Cereal Chemists. Approved Methods of The ABCC. Method 02-51. St. Paus, MN. 1976, Asada, K., Tanaka, K. and Kasai, Z. Cereal Grains. Ann NY Acad i 165:601-814, 1969. Averill, H. P., and King, C. G. The Pbytin Content of Foodstuffs. J. Am. Chem. Soc. 48724-728, 1926. Bassiri, A. and Wahapetian, A. Differences in Concentration and. interrelationships of Phytate, 2hosphorcus. Magnesium, pate, hore Dryland and Irrigated Conditions. J. Agric. Food Chem. 25:1118-1122, 1977. Bruce, H. M. and Callow, R. K. Cereals and Rickets, The Role of Inositol-fexaphosphoric Acid. Biochem J. 28:517-528, 1934. Davis, N. T., Hristic, V., and Flett, A. A. Phytate Rather Than ‘to Rats. Nutr. Rep. Int. 15:207-214, 1977. DeLange, D. J., Joubert, C. Ps, and DuPreeg, S. F. M. The i |» Proc. Nutr. Soc. South, AFR. 2169-76, 1961. Earley, E. B. Determining Phytin Phosphorus. Ind. and Eng. Chem. (anal. Bd.) 16:389-391, 1944. Ellis, R. and Morris, E. R. Comparison of lon-Exchange and Iron Precipitation Methods for Analysis of Phytate. Cereal Chem. 59:232-233, 1982. Franz, K. B., Kennedy, B. M., and Fellers, D. A. Relative Bio- Using-Rat Growth. J. Nutr. 110:2272-2283, 1980. Baghshenas, M., Mahloudji, M., Reinhold, J. C. and Mchamadi, N. i » Am. J. of Clin. Nutr. 25:1143- 146, 1972. Sarlend, B. F. and Oberleas, D. A Modified Method for Phytate Snelysis Using an lon-Exchance Procedure : Textured Megetable Proteins. Cereal Chem, 54:827-832, 1977.Harland, B, F. and Harland, 3. i 4 - Cereal Chem. 57:226- 229, 1980. Hartman, G. H. Removal of Thytate from Soy Protein, J. Am. Oil Chen. Soc. 56:721-735, 1979. Beuber, W. and Stadler, Be i ‘Bestinming Des Phyting. Biochem. 2 6. ’s cited in Chemical Abstracts 8:3569. 122-437, 2934. Hoff~Jorgensen, E., Anderson, 0., and Nielsen, G. The Effect of Biochen. J. 40:555-557, 1946. Johnson, L. F. and Tate, M. &. Structure of "Phytic Beids". Can J, Chem, 47:63-73, 1969. Fnorr, D., Watkins, T. R., and Carlson, B. L. Engymatic Reduction 1 - J. Food Sci, 46:1866- 1869, 1981, Latta, M. and Eskin, M. A Simple and Rapid Colorimetric Method for Poyhate Detemmination. J. Agric. Food Che. 28:1313-1315, 1980. Lim, P. E, and Tate, M. E. i Biochim. Biophys. Acta 250:155- 164, 1971. Biophys Acta 402/516-328, 1973, Lolas, G. Mer Palamidis, N. and Markakis, P. ‘The Phytic Acid-Tota, heat. Cereal Chen, 53:867-871, 1976. ukcitional Significance and Methoss of snaiyera, J Agric. Pood Chem, 30:1-9, 1982. Makower, RU. i i {Zhaseolus wulearis). Cereal Chem 47;288-295, 1970.Marrese, R. Je, Dwell, R. W. and Sprague, M, A. A Comparison of Phosphorus. Cro Sci. 1:80-81, 1962. Mihailovic, M. L., Antic, NM. and Hadzijev, D. Chemical x 8 fous fom Wheat Grain. Plant and Soil 23:117-128, 1965. Nagai, Y. and Funahasi, S. Phytase (tvoinositothexaphophate Phosphohydrolsse) from Wheat Bran Pari 1. Purification and_Substrate Specificity. Agr. Biol. Chem, 26:794-803, 1962. Nahapetian, A. and Bassiri, A. Changes in Concentrations and Inter- i a. Agcic. Food Chen. 23211791182, 1975. Th Hethods of Biochenical Rralysis. Gaited by D. Glick. Vol 20:87-101. New York: Wiley, 1971. Oberleas, D. University of Kentucky, Department of Food Science, Lexington, Kentucky. Personal communication, 1982. Oberleas, D. Muhrer, M. E., and O'Dell, B. L. Dietary Metal- J. utr. 9056-62, 1566. " O'Dell, B. Le, BUEpO, C. E. and Savage, J. EB. Evaluation of Zinc Mute. 1022653-660, 1972. . O'Dell, B. L. and Savace, J. E. Effect of Phytic Acid on zinc Availability. Proc. Soc, Exp. Biol, Med. 103:304-305, 1960. Peers, F. G. The Fhytase of Wheat. Biochem. J, 53:102-110, 1953. Pringle, W. J, S. and Moran, T. Baking. J. Soc. Chem. Ind. 61:108-110, 1942. Reinhold, J. G. Wheat Meals. J, An. Diet, Assoc. 66:38-41, 1975. Reinhold, J. G., Faradji, B., Abadi, P. and Ismail-Beigi, F. Shostughien aj heat Breed yor. 106 :435~ 503, 1976.Samotus, B. and Schwimmer, S. Indirect Method for Determination of Bhytic Acid in Plant Betracts Containing Reducing ‘Substances. Biochim. Biophys. Acta, 57:269-381, 1962. ‘Tangkongchitr, U., Seib, P. A, and Hoseney, R. C. Ibs Fate During BreaGmaking. Cereal Chem. 58:229-234, 1981. ‘Tangkorgchitr, U., Seiby P. A. and foseney, R. C. Phytic Acid TIL. Two_Barriers to the Less of Fiytate Quring Breadmaking. Cereal Chem. 59:216-221, 1962. ‘Ter-Sarkissian, N., Azat, M., Ghavifekr, H., Ferguson, T. and Hedayat, H. High Phytic Acid in Iranian Breads. J. Am. Diet. Assoc. 65:651-653, 1974, U. §. Department of Agriculture, The Official United States Standards for Grain. Washington, D.C.: Government Printing Office, 1978. Wheeler, B. L. and Ferrel, R. BE. 48:312-320, 1971. Williams, S. G. The Role of Phytis Acid in the wheat Grain. Plant Physiol 45:376-381, 1970. Witmer, J. 0. The Bioavailabity of Zinc in Whole Wheat. Master's ‘Thesis, Brighan Young University, 1961- Young, L. x, The Determination of Phytic Acid. Biochem J. 30:252-257, 1936.PHYTIC ACID REDUCTION IN WHOLE WHEAT FLOUR COUGHS BY pH ADJUSINENT OR WITH SPROUTED WHEAT ADDITION Richard Alan Mayfield Department of Food Science and Nutrition M.S. Degree, Decenber 1982 ABSTRACT The reduction of phytic acid in whole wheat doughs was studied using two cultivars of wheat and two methods at three reaction times. The methods were adjustment of dough pi to 5.2, the optimm PH of phytase, with acetic or citric aciés and addition of 18 24-hour or 48-hour sprouted wheats. The mixed doughs were allowed to react either zero, two or four hours. The addition of the acids and a reactien time of two hours resulted in a reduction of phytic acid by approximately 70%. The addition of 24 or 48-hour sprouts did not increase the reduction of phytic acid above the expected reduction ve to natural phytase present in the flour. The rate of reduction slowed significantiy aftec two hours of reaction time for both methods and statistically no difference was noted for two or four hours of reaction tines. COMMITTEE APPROVAL: